CN105353131A - Cytokine multiple detection method based on dual coding and monomolecular counting - Google Patents
Cytokine multiple detection method based on dual coding and monomolecular counting Download PDFInfo
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Abstract
The invention discloses a cytokine multiple detection method based on dual coding and monomolecular counting. The method is characterized in that a first antibody is fixed on a same glass substrate, through interaction of antigen-antibodies, the target objects can be respectively fixed; simultaneously, a secondary antibody and a first coding strand are modified on magnetic nano-particles to obtain a magnetic nano probe; then specific binding effect between the secondary antibody and antigen is used, the magnetic nano probe is fixed on a substrate to form an immune complex having a sandwich structure, the first coding strand on the magnetic nano probe is taken as an amplification unit to trigger a multi-branches hybridization chain reaction, and a double-chain structure having multiple branches; the branched chain is a secondary coding strand; finally, the secondary coding strand and a polymolecule-labeled fluorescence probe are combined, an enhanced fluorescence signal is generated, and quantification on the target object is carried out by counting the amount of phosphor dots. The method realizes multiple and sensitive detection for cytokine, and a detection limit of two target objects is 5fM.
Description
Technical field
The present invention relates to a kind of cell factor multiple detection method based on dual coding and monomolecular counting.
Background technology
Cell factor is the class low molecular protein secreted by immunocyte, in immune response, usually play the effect of signal transduction.Recently, research finds that the existence of some cytokine mediated signal paths and tumour cell, invasion are relevant with transfer, and therefore these cell factors are regarded the biomarker of cancer diagnosis and treatment.But, because a kind of cancer may have multiple mark or the corresponding kinds cancer of a kind of mark possibility, therefore utilize multiple biomarker to detect cancer more accurately feasible.
Conventional cytokines measurement method is Enzyme-linked Immunosorbent Assay test (ELISA), and the method needs first to be fixed on specific antibody by cell factor, then utilizes two of enzyme or fluorophore mark anti-ly to detect it.But because the ratio of object in the method and signal is 1:1, its sensitivity is still restricted.In addition, for Multiple detection, this method also has shortcomings such as easily producing false positive signal, spectra overlapping, cancellation efficiency heterogeneity and instrument requirements complexity.In order to address these problems, the microballoon that polymolecular marks, nano wire, bar code strategy and cross chain reaction etc. are used to the sensitivity improving cytokines measurement.Wherein, bar code strategy has very high amplification efficiency, has been widely used in the Sensitive Detection of biomarker.
Bar code is analyzed as a kind of sensitive analysis strategy, is a kind of by the bioanalytical method of short oligonucleotide sequence object as an alternative.Traditional bar code analysis generally includes magnetic nano particle and metal nanoparticle, and they have all modified recognition unit, therefore can both identify object and form sandwich structure.In addition, metal nanoparticle has also been modified a large amount of oligonucleotide sequences, i.e. bar code chain.Due to the inrichment of nano particle, a small amount of target sequence can be converted into a large amount of bar code chain, and therefore this method has very high sensitivity usually.Based on bar code amplification strategy, Mirkin seminar constructs the multiple detection method of a protide cancer markers, and detectability reaches 170pM.But because the content of this type of cancer markers in serum is extremely low, its sensitivity still cannot meet the needs of actual detection.In addition, the method needs bar code chain to discharge from nano particle, adds the complicacy of detection.
Summary of the invention
Object of the present invention is exactly to solve the problem, and provides a kind of cell factor multiple detection method based on dual coding and monomolecular counting.
To achieve these goals, the present invention adopts following technical scheme:
Based on a cell factor multiple detection method for dual coding and monomolecular counting, step is as follows: the antibody of object is fixed in the substrate of glass of silanization by (1); (2) object and magnetic nano-probe are joined the immune complex above-mentioned substrate of glass being formed sandwich style successively, described magnetic nano-probe is by anti-by two and oligonucleotides-modified obtained to streptavidin-MNBs, and described oligonucleotide chain is as one-level bar code chain; (3) above-mentioned one-level bar code chain triggers the cross chain reaction of multi-branched as amplification original paper, produces the long duplex structure with Duo Tiao branch; (4) fluorescence probe that the long duplex structure described in marks as secondary bar code chain and polymolecular is combined, and produces fluorescence; (5) counted by several point.
Concrete: a kind of cell factor multiple detection method based on dual coding and monomolecular counting, step is as follows:
First testing sample (preferred serum) is added in the substrate of corresponding antibodies bag quilt, hatch 1-3h (preferred 2h) for 37 DEG C, after utilizing PBS-T and PBS to clean respectively, add magnetic nano-probe, 37 DEG C hatch 1-3h (preferred 2h) after, remove unreacted magnetic nano-probe; Then, add hairpin structure potpourri, with HCR damping fluid, hatch 2-4h (preferred 3h), after utilizing PBS-T and PBS to clean respectively, add fluorescence probe for 37 DEG C, hatch 3-5h (preferred 4h) for 37 DEG C, finally utilize PBS-T and PBS to clean respectively, add PBS, under inverted microscope, carry out imaging.
Preferred: antibody is by the preparation of matrix: night incubation at adding antibody 37 DEG C to the substrate of glass of silanization; Then, add mass concentration (w/v) 5% (to get 5 μ gBSA powder join in 100 μ LPBS damping fluids and get final product.) BSA (bovine serum albumin) solution at 37 DEG C close 5-8h (preferred 6h).
Preferred: the preparation of magnetic nano-probe: first, under the effect of externally-applied magnetic field, streptavidin-MNBs is joined in TTL damping fluid and clean, adding PBS after having cleaned makes it suspend, and gets streptavidin-MNBs solution and adds biotinylated bar code chain (1-bio-strand and 2-bio-strand, sequence is in table 1), react at 37 DEG C, by the suspending liquid that obtains under the effect of externally-applied magnetic field, be magnetic nano-probe with final product after PBS cleaning, it is for subsequent use to add PBS;
Preferred: the assembling of polymolecular mark fluorescent probe: first by three fluorescently-labeled single stranded DNA (1-Ca, 1-Cb, 1-Cc or 2-Ca, 2-Cb, 2-Cc sequence is in table 1) and a long single stranded DNA (1-Cd or 2-Cd sequence is in table 1) respectively with the dilution of TE damping fluid; Then respectively each single stranded DNA is added in TM damping fluid, mixing; Then potpourri is put into the metal bath of 95 DEG C, annealing or quenching, obtained fluorescence probe.
Above-mentioned method is applied to non-diagnostic object vitro detection sample.
A kind of cytokine detection kits based on dual coding and monomolecular counting, it is characterized in that: comprise the corresponding primary antibodie of detected cell factor, the substrate of glass of silanization, magnetic nano-probe, it is anti-and oligonucleotides-modified that described magnetic nano-probe is detected cell factor corresponding two, hairpin structure DNA, described hairpin structure DNA has the base sequence matched with the oligonucleotides of described magnetic nano-probe, and pairing forms the long duplex structure with Duo Tiao branch, fluorescence probe, described fluorescence probe hybridizes by four single stranded DNAs the decussate texture produced, its one end is the overhanging sequence stretched out, other three ends all marked fluorophor, described overhanging sequence is combined with the long duplex structure with Duo Tiao branch.
Beneficial effect of the present invention:
Bar code analysis and monomolecular counting combine for the detection of protein by method of the present invention first, and the sensitivity of method is 5fM, and detect while can realizing in complex samples two kinds and two or more object.In addition, we analyze human serum sample, illustrate that this method is reliable, have very large application potential in clinical detection.
The method does not need bar code chain to discharge from nano particle, and step is simple.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the inventive method;
Fig. 2 is the gel electrophoresis analysis of fluorescence probe, 1-5 band is the electrophoretic image of quenching product, 6-10 is by the electrophoretic image of annealed product, and be 1-Cd, 1-Cd+1-Cc, 1-Cd+1-Cc+1-Cb, 1-Cd+1-Cc+1-Ca and 1-Cd+1-Cc+1-Cb+1-Ca respectively, M is marker;
Fig. 3 is that fluorescence is counted and the linear relationship chart of target concentration;
Fig. 4 is the fluorescence response of different target thing, and 1 is bovine serum albumin(BSA), and 2 is fibrin ferments, and 3 is IgG, and 4 is that first tire egg 6 is white, and 5 is carcinomebryonic antigens, and 6 is IFN-γ and TNF-α.
Embodiment
Reagent and material huamn tumor necrosis factory alpha (TNF-α), recombinant human interferon gamma (IFN-γ), tumor necrosis factor α antibody (anti-TNF-α), biotinylated tumor necrosis factor-alpha antibody (bio-anti-TNF-α), interferon gamma antibody (anti-IFN-γ), biotinylated interferon gamma antibody (bio-anti-IFN-γ) and human IgG are purchased from Abcam company.The magnetic ball (streptavidin-MNBs, 350nm) of streptavidin is purchased from BangsLaboratories company.Bovine serum albumin(BSA) (BSA) is purchased from Shanghai Sheng Gong bioengineering company limited (Shanghai).The all solution used in experiment are all prepared by ultrapure water, and all nucleotide sequences synthesize (Beijing) by Ying Weijie base company limited.
Instrument epifluorescence microscope system (OlympusOpaticalCo.Ltd, Tokyo, Japan), mainly comprise inverted microscope (OlympusIX81), Halogen lamp LED (OlympusLG-PS2), inverted fluorescence microscope control module (OlympusIX2-UCB), the multistage amplification inductive coupler of electronics (ElectronMultiplyingChargeCoupledDevice, EMCCD, Cascade512B, RoperScientific, Tuscon, AZ.USA), MetaMorph software systems (UniversalImaging, Downingtown, PA, USA), precision optics vibration-isolating platform (Yiao Information Optical Science & Technology Co., Ltd., Shanghai, Shanghai), supersonic cleaning machine (Branson-200 type, sino-america joint-venture Bi Nengxinchao company limited, Shanghai), DHG-9070A type electric heating constant-temperature blowing drying box (the upper grand experimental facilities company limited of Nereid, Shanghai), DNP-9052 type electro-heating standing-temperature cultivator (the upper grand experimental facilities company limited of Nereid, Shanghai).
First antibody bag is prepared the substrate of glass of silanization by the preparation of substrate, then basadly adds 50 μ L corresponding antibodies (anti-IFN-γ andanti-TNF-α, 0.01nM) night incubation.Then, the BSA solution adding 5% closes 6h at 37 DEG C.Each all will use PBS-T (0.15MNaCl, 7.6mMNa after having walked respectively
2hPO
4, 2.4mMNaH
2pO
4, 0.05% Tween-20, PH7.4) and PBS (0.15MNaCl, 7.6mMNa
2hPO
4, 2.4mMNaH
2pO
4, pH7.4) and buffer solution for cleaning three times, to go out unreacted reagent.
First 2 μ Lstreptavidin-MNBs, under the effect of externally-applied magnetic field, join in 200 μ LTTL damping fluids (100mMTris, 1MLiCl, 0.1%Tween-20, pH8.0) and clean 5 times by the preparation of magnetic nano-probe.Adding 60 μ LPBS after having cleaned makes it suspend.Then, get 30 μ Lstreptavidin-MNBs solution and add the biotinylated bar code chain of 10 μ L (1-bio-strand and 2-bio-strand, nucleotide sequence is in table 1), at 37 DEG C, reacting 5h.Then, by the suspending liquid that obtains under the effect of externally-applied magnetic field, three times are cleaned with PBS.Final product is magnetic nano-probe, and it is for subsequent use to add 50 μ LPBS.
The assembling of polymolecular mark fluorescent probe and electrophoresis checking are first by three fluorescently-labeled single stranded DNA (1-Ca, 1-Cb, 1-Ccor2-Ca, 2-Cb, 2-Cc, nucleotide sequence is in table 1) and a long single stranded DNA (1-Cd or 2-Cd, nucleotide sequence is in table 1) use TE damping fluid (10mMTris, 1mMNa respectively
2eDTA, pH8.0) be diluted to 10 μMs.Then get each single stranded DNA of 10 μ L respectively and add 60 μ LTM damping fluid (20mMTris, 50mMMgCl
2, pH8.0) in, mixing.Then potpourri is put into the metal bath of 95 DEG C, be down to 30 DEG C (annealing) in 2min or put into the frozen water (quenching) of 4 DEG C at once, then obtain the fluorescence probe that two kinds of methods produce, its final concentration is 0.1 μM.
In order to verify the formation of this fluorescent nano probe and select optimum synthetic method, we utilize the polyacrylamide (native-PAGE) of 12% to carry out electrophoresis experiment experiment to it.
50 μ L objects (IFN-γ and TNF-α) first add in the substrate of antibody bag quilt by the structure based on the multiple analysis method of two bar code strategy and monomolecular counting, hatch 2h for 37 DEG C.After utilizing PBS-T and PBS to clean three times respectively, add 50 μ L magnetic nano-probes.37 DEG C hatch 2h after, utilize magnetic separation technique to be washed away by unreacted magnetic nano-probe.Then, add 50 μ L hairpin structure potpourris (nucleotide sequence of each hairpin structure is in table 1), comprise 10 μ LH1 (1-H1 and 2-H1,0.2 μM), 10 μ LH2 (1-H2 and 2-H2,0.22 μM) and 30 μ LHCR damping fluid (50mMNa
2hPO
4, 0.5NaCl, pH6.8), hatch 4h at 37 DEG C.After utilizing PBS-T and PBS to clean three times respectively, add 50 μ L fluorescence probes, at 37 DEG C, hatch 4h.Finally utilize PBS-T and PBS to clean three times respectively, add 50PBS, under inverted microscope, carry out imaging.
The antibody of object IFN-γ and TNF-α, as Fig. 1, first, by epoxy radicals-hydroxyl reaction, is fixed in the substrate of glass of silanization by principle, meanwhile, prepares magnetic nano-probe by resisting two with oligonucleotides-modified to streptavidin-MNBs.Wherein, the effect of two anti-performance molecular recognition, oligonucleotide chain is one-level bar code chain.Then, object and magnetic nano-probe are joined in substrate successively, formed the immune complex of sandwich style by the immune response between antigen-antibody.Next, one-level bar code chain triggers the cross chain reaction (mHCR) of multi-branched as amplification original paper, produces the long duplex structure with Duo Tiao branch.The fluorescence probe that this branch can mark with polymolecular as secondary bar code chain is combined, and produces fluorescence.Count finally by inverted microscope number point.
The assembling of polymolecular fluorescence probe and sign
Inventor has devised the novel fluorescence probe that is marked with three fluorescence molecules, become the fluorescence probe of polymolecular mark.This probe hybridizes by four single stranded DNAs the decussate texture produced, and its one end is the overhanging sequence stretched out, and other three ends all marked fluorophor.
Inventor have selected two kinds of methods, namely anneals and quenches and synthesize this probe respectively, in order to characterization probes successful synthesis and select the best approach, We conducted electrophoresis experiment.As Fig. 2,5 and 10 bands are by annealing and the fluorescence probe obtained that quenches respectively, and as can be seen from the figure, the probe band that the method for annealing obtains is brighter, illustrates that productive rate is higher.
Condition optimizing
Inventor is optimized the ratio of BSA off-period, level encoder chain concentration, H1 concentration, H2 and H1 and fluorescence probe incubation time respectively.Final selection BSA off-period is 6h, and level encoder chain concentration is 0.2 μM, and H1 concentration is 0.2 μM, and H2/H1 is 1.1, and fluorescence probe incubation time is 4h.
Method validation
In optimal conditions, first inventor investigates the sensitivity of method, and as Fig. 3, within the scope of 5fM-100fM, fluorescence is counted good with the linear relationship of object, and linear equation is respectively Y
1=6.207+7.541C
1and Y
2=5.034+7.555C
2, 5fM is to the detectability of two kinds of objects.
Then, the specificity of inventor to method is investigated, and as Fig. 4, the method only has stronger fluorescence response to object, less to the fluorescence response of other object, illustrates that this method has good specificity.
Finally, inventor's profit is analyzed human serum in this way, and as table 2, the amount obtaining IFN-γ and TNF-α in human serum is 1.04-1.07 × 10 respectively
-13mol/L and 3.29-5.26 × 10
-13mol/L, what achieve two kinds of objects in serum is quantitative.
Nucleotide sequence used by table 1
Table 2 human serum quantitatively detects
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (10)
1., based on a cell factor multiple detection method for dual coding and monomolecular counting, it is characterized in that: step is as follows: the antibody of object is fixed in the substrate of glass of silanization by (1); (2) substrate of glass object and magnetic nano-probe being joined successively step (1) is formed the immune complex of sandwich style, described magnetic nano-probe is by anti-by two and oligonucleotides-modified obtained to streptavidin-MNBs, and described oligonucleotide chain is as one-level bar code chain; (3) the one-level bar code chain of step (2) triggers the cross chain reaction of multi-branched as amplification original paper, produces the long duplex structure with Duo Tiao branch; (4) fluorescence probe that the long duplex structure of step (3) marks as secondary bar code chain and polymolecular is combined, and produces fluorescence; (5) counted by several point.
2., based on a cell factor multiple detection method for dual coding and monomolecular counting, it is characterized in that: step is as follows:
(1) first testing sample is added in the substrate of corresponding antibodies bag quilt, hatch 2h for 37 DEG C; (2), after utilizing PBS-T and PBS to clean respectively, add magnetic nano-probe, 37 DEG C hatch 2h after, form the immune complex of sandwich style, remove unreacted magnetic nano-probe, described magnetic nano-probe has the modification of corresponding two anti-and oligonucleotides; (3) add hairpin structure potpourri and HCR damping fluid, hatch 4h for 37 DEG C, the oligonucleotides on described hairpin structure and magnetic nano-probe matches the long duplex structure formed with Duo Tiao branch; (4) after utilizing PBS-T and PBS to clean respectively, add fluorescence probe, hatch 4h for 37 DEG C, described fluorescence probe hybridizes by four single stranded DNAs the decussate texture produced, its one end is the overhanging sequence stretched out, other three ends all marked fluorophor, and described overhanging sequence is combined with the long duplex structure with Duo Tiao branch; (5) utilize PBS-T and PBS to clean respectively, add PBS, counted by inverted microscope number point.
3. detection method as claimed in claim 2, is characterized in that: described testing sample is blood serum sample.
4. detection method as claimed in claim 2, it is characterized in that: described fluorescence probe is respectively with the dilution of TE damping fluid by three fluorescently-labeled single stranded DNAs and a long single stranded DNA, then respectively each single stranded DNA is added in TM damping fluid, mixing, then potpourri is put into the metal bath of 95 DEG C, annealing or quenching obtain.
5. detection method as claimed in claim 4, is characterized in that: described three fluorescently-labeled single stranded sequences are: the sequence of SEQIDNO:14, SEQIDNO:14, SEQIDNO:14 or SEQIDNO:14, SEQIDNO:14, SEQIDNO:14 and a long single stranded DNA is: SEQIDNO:14 or SEQIDNO:14.
6. detection method as claimed in claim 2, is characterized in that: in described step (1), antibody bag is as follows by the preparation method of matrix: night incubation at adding antibody 37 DEG C to the substrate of glass of silanization; Then, BSA solution closed 5-8h at 37 DEG C that mass concentration is 5% is added.
7. detection method as claimed in claim 6, is characterized in that: add BSA solution closed 6h at 37 DEG C that mass concentration is 5%.
8. detection method as claimed in claim 2, it is characterized in that: the preparation method of magnetic nano-probe in described step (2): first, under the effect of externally-applied magnetic field, streptavidin-MNBs is joined in TTL damping fluid and clean, adding PBS after having cleaned makes it suspend, and gets streptavidin-MNBs solution and adds biotinylated bar code chain, reacts at 37 DEG C, by the suspending liquid that obtains under the effect of externally-applied magnetic field, be magnetic nano-probe with final product after PBS cleaning.
9. detection method as claimed in claim 8, is characterized in that: the sequence of described biotinylated bar code chain is: SEQIDNO:1 and SEQIDNO:2.
10. the cytokine detection kits based on dual coding and monomolecular counting, it is characterized in that: comprise the corresponding primary antibodie of detected cell factor, the substrate of glass of silanization, magnetic nano-probe, it is anti-and oligonucleotides-modified that described magnetic nano-probe is detected cell factor corresponding two, hairpin structure DNA, described hairpin structure DNA has the base sequence matched with the oligonucleotides of described magnetic nano-probe, and pairing forms the long duplex structure with Duo Tiao branch, fluorescence probe, described fluorescence probe hybridizes by four single stranded DNAs the decussate texture produced, its one end is the overhanging sequence stretched out, other three ends all marked fluorophor, described overhanging sequence is combined with the long duplex structure with Duo Tiao branch.
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| JP2020516232A (en) * | 2016-12-16 | 2020-06-11 | アラトム， エルエルシー | Molecular detection using ligation amplification |
| JP7093355B2 (en) | 2016-12-16 | 2022-06-29 | アラトム， エルエルシー | Molecule detection using ligation amplification |
| CN108753941A (en) * | 2018-06-22 | 2018-11-06 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Double labeling magnetic bead and its preparation method and application |
| CN110940809A (en) * | 2019-12-09 | 2020-03-31 | 福州大学 | Technology for detecting alpha-fetoprotein in blood based on combination of laser-induced fluorescence and paper chip |
| CN112964868A (en) * | 2021-02-05 | 2021-06-15 | 华中农业大学 | Biochemical analysis method for simultaneously detecting multiple target objects based on magnetic separation |
| CN112964868B (en) * | 2021-02-05 | 2022-06-28 | 华中农业大学 | Biochemical analysis method for simultaneously detecting multiple target objects based on magnetic separation |
| CN114277098A (en) * | 2021-09-27 | 2022-04-05 | 清华大学 | Bar code system based on DNA paper folding structure |
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